This application relates generally to chimeric heteromultimer adhesins comprising extracellular binding domains of heteromultimeric receptors, which heteromultimer adhesins bind the ligand of the natural receptor. The invention further relates to antibodies to the heteroadhesins, methods of making the adhesins and methods of using the heteroadhesins and antibodies.
Transduction of signals that regulate cell growth and differentiation is regulated in part by phosphorylation of various cellular proteins. Protein tyrosine kinases are enzymes that catalyze this process. Receptor protein tyrosine kinases are believed to direct cellular growth via ligand-stimulated tyrosine phosphorylation of intracellular substrates.
The ErbB family of single-spanning, receptor tyrosine kinases consists of four members: epidermal growth factor receptor (EGFR), ErbB2 (HER2/neu), ErbB3 (HER3) and ErbB4 (HER4). A number of ligands, all of which are different gene products, have been identified that bind and activate EGFR (reviewed in Groenen et al., 1994). In contrast, a single neuregulin gene encodes for a large number of protein isoforms that result from alternative splicing of mRNA transcripts (reviewed in (Lemke, G. (1996) mol. Cell. Neurosci. 7:247-262). ErbB3 (Carraway, K. L. et al. (1994) J. Biol. Chem. 269:14303-14306) or ErbB4 (Plowman, G. D. et al., (1993) Nature 366:473-475) can serve as receptors for the neuregulins. These receptors and ligands play key roles in normal cell growth and differentiation.
Growth factor receptor protein tyrosine kinases of the class I subfamily include the 170 kDa epidermal growth factor receptor (EGFR) encoded by the erbB1 gene. erbB1 has been causally implicated in human malignancy. In particular, increased expression of this gene has been observed in more aggressive carcinomas of the breast, bladder, lung and stomach (Modjtahedi, H. and Dean, C. (1994) Int. J. Oncol. 4:277-296).
The second member of the class I subfamily, p185neu, was originally identified as the product of the transforming gene from neuroblastomas of chemically treated rats. The neu gene (also called erbB2 and HER2) encodes a 185 kDa receptor protein tyrosine kinase. Amplification and/or overexpression of the human HER2 gene correlates with a poor prognosis in breast and ovarian cancers (Slamon, D. J. et al., Science 235:177-182 (1987); and Slamon et al., Science 244:707-712 (1989)). Overexpression of HER2 has been correlated with other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon and bladder. Accordingly, Slamon et al. in U.S. Pat. No. 4,968,603 describe and claim various diagnostic assays for determining HER2 gene amplification or expression in tumor cells. Slamon et al. discovered that the presence of multiple gene copies of HER2 oncogene in tumor cells indicates that the disease is more likely to spread beyond the primary tumor site, and that the disease may therefore require more aggressive treatment than might otherwise be indicated by other diagnostic factors. Slamon et al. conclude that the HER2 gene amplification test, together with the determination of lymph node status, provides greatly improved prognostic utility.
A further related gene, called erbB3 or HER3, has also been described. See U.S. Pat. No. 5,183,884; Kraus et al., Proc. Natl. Acad. Sci. USA 86:9193-9197 (1989); EP Pat Appln No 444,961A1; and Kraus et al., Proc. Natl. Acad. Sci. USA 90:2900-2904 (1993). Kraus et al. (1989) discovered that markedly elevated levels of erbB3 mRNA were present in certain human mammary tumor cell lines indicating that erbB3, like erbB1 and erbB2, may play a role in human malignancies. Also, Kraus et al. (1993) showed that EGF-dependent activation of the ErbB3 catalytic domain of a chimeric EGFR/ErbB3 receptor resulted in a proliferative response in transfected NIH-3T3 cells. Furthermore, these researchers demonstrated that some human mammary tumor cell lines display a significant elevation of steady-state ErbB3 tyrosine phosphorylation further indicating that this receptor may play a role in human malignancies. The role of erbB3 in cancer has been explored by others. It has been found to be overexpressed in breast (Lemoine et al., Br. J. Cancer 66:1116-1121 (1992)), gastrointestinal (Poller et al., J. Pathol. 168:275-280 (1992), Rajkumer et al., J. Pathol. 170:271-278 (1993), and Sanidas et al., Int. J. Cancer 54:935-940 (1993)), and pancreatic cancers (Lemoine et al., J. Pathol. 168:269-273 (1992), and Friess et al., Clinical Cancer Research 1:1413-1420 (1995)).
ErbB3 is unique among the ErbB receptor family in that it possesses little or no intrinsic tyrosine kinase activity (Guy et al., Proc. Natl. Acad. Sci. USA 91:8132-8136 (1994) and Kim et al. J. Biol. Chem. 269:24747-55 (1994)). When ErbB3 is co-expressed with ErbB2, an active signaling complex is formed and antibodies directed against ErbB2 are capable of disrupting this complex (Sliwkowski et al., J. Biol. Chem., 269(20):14661-14665 (1994)). Additionally, the affinity of ErbB3 for heregulin (HRG) is increased to a higher affinity state when co-expressed with ErbB2. See also, Levi et al., Journal of Neuroscience 15: 1329-1340 (1995); Morrissey et al., Proc. Natl. Acad. Sci. USA 92: 1431-1435 (1995); Lewis, G. D. et al., Cancer Res., 56:1457-1465 (1996); Pinkas-Kramarski, R. et al. (1996) EMBO J. 15:2452-2467; Beerli, R. et al. (1995) Mol. Cell. Biol. 15:6496-6505; and Karunagaran, D. et al. (1996) EMBO J. 15:254-264 with respect to the in vivo ErbB2-ErbB3 protein complex.
The class I subfamily of growth factor receptor protein tyrosine kinases has been further extended to include the HER4/Erb4 receptor. See EP Pat Appln No 599,274; Plowman et al., Proc. Natl. Acad. Sci. USA 90:1746-1750 (1993); and Plowman et al., Nature 366:473-475 (1993). Plowman et al. found that increased HER4 expression closely correlated with certain carcinomas of epithelial origin, including breast adenocarcinomas. Diagnostic methods for detection of human neoplastic conditions (especially breast cancers) which evaluate HER4 expression are described in EP Pat Appln No. 599,274.
The quest for the activator of the HER2 oncogene has lead to the discovery of a family of heregulin polypeptides. These proteins appear to result from alternate splicing of a single gene which was mapped to the short arm of human chromosome 8 by Lee, J. and Wood, W. I. (1993) Genomics 16:790-791).
Holmes et al. isolated and cloned a family of polypeptide activators for the HER2 receptor which they called heregulin-xcex1 (HRG-xcex1), heregulin-xcex21 (HRG-xcex21), heregulin-xcex22 (HRG-xcex22), heregulin-xcex22-like (HRG-xcex22-like), and heregulin-xcex23 (HRG-xcex23). See Holmes, W. E. et al., Science 256:1205-1210 (1992); WO 92/20798; and U.S. Pat. No. 5,367,060. The 45 kDa polypeptide, HRG-xcex1, was purified from the conditioned medium of the MDA-MB-231 human breast cancer cell line. These researchers demonstrated the ability of the purified heregulin polypeptides to activate tyrosine phosphorylation of the HER2 receptor in MCF7 breast tumor cells. Furthermore, the mitogenic activity of the heregulin polypeptides on SK-BR-3 cells (which express high levels of the HER2 receptor) was illustrated. Like other growth factors which belong to the EGF family, soluble HRG polypeptides appear to be derived from a membrane bound precursor (called pro-HRG) which is proteolytically processed to release the 45 kDa soluble form. These pro-HRGs lack a N-terminal signal peptide.
While heregulins are substantially identical in the first 213 amino acid residues, they are classified into two major types, xcex1 and xcex2, based on two variant EGF-like domains which differ in their C-terminal portions. Nevertheless, these EGF-like domains are identical in the spacing of six cysteine residues contained therein. Based on an amino acid sequence comparison, Holmes et al. found that between the first and sixth cysteines in the EGF-like domain, HRGs were 45% similar to heparin-binding EGF-like growth factor (HB-EGF), 35% identical to amphiregulin (AR), 32% identical to TGF-xcex1, and 27% identical to EGF.
The 44 kDa neu differentiation factor (NDF), which is the rat equivalent of human HRG, was first described by Peles et al., Cell, 69:205-216 (1992); and Wen et al., Cell, 69:559-572 (1992). Like the HRG polypeptides, NDF has an immunoglobulin (Ig) homology domain followed by an EGF-like domain and lacks a N-terminal signal peptide. Subsequently, Wen et al., Mol. Cell. Biol., 14(3):1909-1919 (1994) carried out xe2x80x9cexhaustive cloningxe2x80x9d to extend the family of NDFs. This work revealed six distinct fibroblastic pro-NDFs. Adopting the nomenclature of Holmes et al., the NDFs are classified as either xcex1 or xcex2 polypeptides based on the sequences of the EGF-like domains. Isoforms 1 to 4 are characterized on the basis of the variable membrane stretch (between the EGF-like domain and transmembrane domain). Also, isoforms a, b and c are described which have variable length cytoplasmic domains. These researchers conclude that different NDF isoforms are generated by alternative splicing and perform distinct tissue-specific functions. See also EP 505 148; WO 93/22424; and WO 94/28133 concerning NDF.
While the heregulin polypeptides were first identified based on their ability to activate the HER2 receptor (see Holmes et al., supra), it was discovered that certain ovarian cells expressing neu and neu-transfected fibroblasts did not bind or crosslink to NDF, nor did they respond to NDF to undergo tyrosine phosphorylation (Peles et al., EMBO J. 12:961-971 (1993)). This indicated another cellular component was necessary for conferring full heregulin responsiveness. Carraway et al. subsequently demonstrated that 125I-rHRGxcex21177-244 bound to NIH-3T3 fibroblasts stably transfected with bovine erbB3 but not to non-transfected parental cells. Accordingly, they conclude that ErbB3 is a receptor for HRG and mediates phosphorylation of intrinsic tyrosine residues as well as phosphorylation of ErbB2 receptor in cells which express both receptors. Carraway et al., J. Biol. Chem. 269(19):14303-14306 (1994). Sliwkowski et al., J. Biol. Chem. 269(20):14661-14665 (1994) found that cells transfected with HER3 alone show low affinities for heregulin, whereas cells transfected with both HER2 and HER3 show higher affinities.
This observation correlates with the xe2x80x9creceptor cross-talkingxe2x80x9d described previously by Kokai et al., Cell 58:287-292 (1989); Stern et al., EMBO J. 7:995-1001 (1988); and King et al., 4:13-18 (1989). These researchers found that binding of EGF to the EGFR resulted in activation of the EGFR kinase domain and cross-phosphorylation of p185HER2. This is believed to be a result of ligand-induced receptor heterodimerization and the concomitant cross-phosphorylation of the receptors within the heterodimer (Wada et al., Cell 61:1339-1347 (1990)).
Plowman and his colleagues have similarly studied p180HER4/p185HER2 activation. They expressed p185HER2 alone, p180HER4 alone, or the two receptors together in human T lymphocytes and demonstrated that heregulin is capable of stimulating tyrosine phosphorylation of p180HER4, but could only stimulate p185HER2 phosphorylation in cells expressing both receptors. Plowman et al., Nature 336:473-475 (1993). Thus, heregulin is an example of a member of the EGF growth factor family that can interact with several receptors (Carraway and Cantley, Cell 78:5-8 (1994)). Additionally, the xcex2-cellulin ligand has been shown to bind to the EGF receptor and HER4, but does not bind HER3 (Riese II, D. J. et al. (1996) Oncogene 12:345-353).
The biological role of heregulin has been investigated by several groups. For example, Falls et al., (Cell 72:801-815 (1993)) found that ARIA plays a role in myotube differentiation, namely affecting the synthesis and concentration of neurotransmitter receptors in the postsynaptic muscle cells of motor neurons. Corfas and Fischbach demonstrated that ARIA also increases the number of sodium channels in chick muscle. Corfas and Fischbach, J. Neuroscience, 13(5): 2118-2125 (1993). It has also been shown that GGFII is mitogenic for subconfluent quiescent human myoblasts and that differentiation of clonal human myoblasts in the continuous presence of GGFII results in greater numbers of myotubes after six days of differentiation (Sklar et al., J. Cell Biochem., Abst. W462, 18D, 540 (1994)). See also WO 94/26298 published Nov. 24, 1994.
Holmes et al., supra, found that HRG exerted a mitogenic effect on mammary cell lines (such as SK-BR-3 and MCF-7). The mitogenic activity of GGFs on Schwann cells has also been reported. See, e.g., Brockes et al., J. Biol. Chem. 255(18):8374-8377 (1980); Lemke and Brockes, J. Neurosci. 4:75-83 (1984); Brockes et al., J. Neuroscience 4(1):75-83 (1984); Brockes et al., Ann. Neurol. 20(3):317-322 (1986); Brockes, J., Methods in Enzym., 147: 217-225 (1987) and Marchionni et al., supra. Schwann cells provide myelin sheathing around the axons of neurons, thereby forming individual nerve fibers. Thus, it is apparent that Schwann cells play an important role in the development, function and regeneration of peripheral nerves. The implications of this from a therapeutic standpoint have been addressed by Levi et al., J. Neuroscience 14(3):1309-1319 (1994). Levi et al. discuss the potential for construction of a cellular prosthesis comprising human Schwann cells which could be transplanted into areas of damaged spinal cord. Methods for culturing Schwann cells ex vivo have been described. See WO 94/00140 and Li et al., J. Neuroscience 16(6):2012-2019 (1996).
Pinkas-Kramarski et al. found that NDF seems to be expressed in neurons and glial cells in embryonic and adult rat brain and primary cultures of rat brain cells, and suggested that it may act as a survival and maturation factor for astrocytes (Pinkas-Kramarski et al., PNAS, USA 91:9387-9391 (1994)). Meyer and Birchmeier, PNAS, USA 91:1064-1068 (1994) analyzed expression of heregulin during mouse embryogenesis and in the perinatal animal using in situ hybridization and RNase protection experiments. These authors conclude that, based on expression of this molecule, heregulin plays a role in vivo as a mesenchymal and neuronal factor. Also, their findings imply that heregulin functions in the development of epithelia. Similarly, Danilenko et al., Abstract 3101, FASEB 8(4-5):A535 (1994), found that the interaction of NDF and the HER2 receptor is important in directing epidermal migration and differentiation during wound repair.
Interaction of ErbB family members has been investigated in vitro and in vivo. Transactivation of ErbB2 as a result of ligand interaction with other ErbB family members is a common and physiologically important occurrence (Dougall, W. C. et al., (1993) J. Cell. Biochem. 53:61-73; Earp, H. S. et al., (1995) Breast Cancer Res. Treatment 35:115-132). Co-expression of ErbB2 with ErbB3 leads to the formation of a high affinity heregulin (HRG) binding site (Sliwkowski, M. X. et al., (1994) J. Biol. Chem. 269:14661-14665). ErbB2 modulates the affinity of ErbB3 for HRG and appears to provide tyrosine kinase activity to the ErbB3-HRG complex, since ErbB3 is a dysfunctional signaling receptor lacking intrinsic tyrosine kinase activity (Guy, P. M. et al., (1994) PNAS USA 91:8132-8136). Physio-chemical studies have not shown association of the ECDs of ErbB2 and ErbB3 in vitro (Horan et al., J. Biol. Chem. 270:24604-24608 (1995)). In addition, binding of neu differentiation factor (NDF) to soluble HER3 was not enhanced by the presence of soluble HER2.
The invention relates to the surprising discovery that soluble chimeric heteromultimers comprising the extracellular domains of heteromultimeric receptor monomers bind the receptor ligand. The invention further relates to methods of making the chimeric heteromultimers, methods of using them as receptor ligand antagonists, antibodies to the chimeric heteromultimers that function as antagonists or agonists of the receptor ligand, and methods of treating a disease state related to ligand-receptor interaction.
In one aspect the invention includes a chimeric heteromultimer comprising a first amino acid sequence, which sequence forms a chimeric monomer and comprises an extracellular domain (ECD) or ligand binding fragment thereof, of a first monomer of a natural heteromultimeric receptor and a multimerization domain, wherein the ECD is fused to the multimerization domain. The chimeric heteroadhesin of the invention further comprises an additional amine acid sequence forming an additional chimeric monomer comprising an extracellular domain of an additional monomer of the natural heteromultimeric receptor and a multimerization domain. According to this aspect of the invention the extracellular domains of the first and additional monomers of the natural heteromultimeric receptor are associated in a cell to form a natural heteromultimeric receptor which is activated upon binding of a ligand, and wherein the soluble chimeric heteromultimer adhesin has 10xe2x88x921 to 106 fold affinity for the ligand relative to a monomer of the natural receptor or a homomultimer of the natural receptor. In a preferred embodiment of the invention the chimeric heteromultimer adhesin is an aqueous soluble adhesin.
In an embodiment of the invention, the chimeric heteromultimer adhesin is an antagonist of the ligand that binds to the extracellular domains of the natural heteromultimeric receptor.
In another embodiment of the invention, the multimerization domain of the first amino acid sequence is capable of interacting with the multimerization domain of each additional amino acid sequence to form a heteromultimer.
In yet another embodiment of the invention the chimeric heteromultimer adhesin comprises a multimerization domain which includes an immunoglobulin region, preferably an immunoglobulin constant region, such as from IgG1, IgG2, IgG3, IgG4, IgM, and IgF.
In still another embodiment of the invention, the chimeric heteroadhesin includes a multimerization domain capable of forming a stable protein-protein interaction. Such protein-protein interaction domains (or multimerization domains) include a leucine zipper, an amino acid sequence comprising a protuberance complementary to an amino acid sequence comprising a hole, a hydrophobic domain, a hydrophilic domain, and an amino acid sequence comprising a free thiol moiety capable of reacting to form an intermolecular disulfide bond with a multimerization domain of an additional amino acid sequence.
A further embodiment of the invention is a chimeric heteromultimer adhesin in which the first amino acid sequence comprising an extracellular domain of the ErbB2 receptor monomer, an additional amino acid sequence comprising an extracellular domain of the ErbB3 receptor monomer, and a multimerization domain of the first and additional amino acids each comprises an immunoglobulin constant region. The multimerization domain provides for the formation of a stable protein-protein interaction between the first and additional amino acid sequences. A preferred ligand of this chimeric heteroadhesin is the ligand, heregulin.
A further embodiment of the invention is a chimeric heteromultimer adhesin in which the first amino acid sequence comprising an extracellular domain of the ErbB2 receptor monomer, an additional amino acid sequence comprising an extracellular domain of the ErbB4 receptor monomer, and a multimerization domain of the first and additional amino acids each comprises an immunoglobulin constant region. The multimerization domain provides for the formation of a stable protein-protein interaction between the first and additional amino acid sequences. A preferred ligand of this chimeric heteroadhesin is the ligand, heregulin.
In another aspect, the invention includes an isolated nucleic acid sequence encoding an amino acid sequence of the chimeric heteromultimer adhesin of the invention.
In other embodiments, the invention provides an isolated nucleic acid molecule encoding the chimeric amino acid sequence of a monomer of the heteromultimer adhesin such as, for example, ErbB2-IgG, ErbB3-IgG, or ErbB4-IgG. For example, the nucleic acid molecule may be selected from the group consisting of: (a) a nucleic acid comprising the nucleotide sequence of the extracellular domain (i.e. a ligand binding domain or binding fragment thereof) of a monomer of a natural heteromultimeric receptor covalently attached in phase and in the direction of transcription to a nucleic acid sequence encoding a multimerization domain, such as an immunoglobulin constant domain; and (b) a nucleic acid corresponding to the sequence of (a) within the scope of degeneracy of the genetic code. The isolated nucleic acid molecule optionally further comprises a promoter operably linked thereto.
The isolated nucleic acid may also be used for in vivo or ex vivo gene therapy. This embodiment of the invention encompasses the administration of the nucleic acid of the invention, a vector comprising the nucleic acid, or a cell comprising the nucleic acid to a mammal such that the encoded chimeric adhesin is expressed in the mammal and acts as an antagonist of its ligand. For example, ErbB2/3-IgG expressed in a mammal is useful to reduce the local concentration of heregulin near a ErbB2/3 receptor and inhibit growth of a cell having the receptor on its surface. Preferably the expressed ErbB2/3-IgG is used to treat a cell proliferative disease, such as a cancer, in which antagonizing heregulin binding to its receptor inhibits cell growth.
In an embodiment of the invention, the isolated nucleic acid sequence of the chimeric amino acid encodes an extracellular domain or binding fragment thereof from the ErbB2 receptor, and wherein the multimerization domain comprises an immunoglobulin constant region.
In still another embodiment of the invention, the isolated nucleic acid sequence of the chimeric amino acid sequence encodes an extracellular domain or binding fragment thereof from the ErbB3 receptor ECD, and wherein the multimerization domain comprises an immunoglobulin constant region.
In another embodiment of the invention, the isolated nucleic acid sequence of the chimeric amino acid sequence encodes an extracellular domain or binding fragment thereof from the ErbB4 receptor ECD, and wherein the multimerization domain comprises an immunoglobulin constant region.
Another embodiment of the invention includes a promoter operably linked to the nucleic acid molecule.
In still another embodiment, the invention includes a vector comprising the isolated nucleic acid of the invention. For example, the invention provides a vector comprising the nucleic acid molecule (e.g. an expression vector comprising the nucleic acid molecule operably linked to control sequences recognized by a host cell transformed with the vector); a host cell comprising the nucleic acid molecule; and a method of using a nucleic acid molecule encoding a chimeric heteromultimer adhesin, such as an ErbB-IgG, to effect production of the adhesin which comprises the step of culturing the host cell and recovering the adhesin from the cell culture. In a related embodiment the method of using the nucleic acid to effect production of the adhesin includes introducing multiple nucleic acid sequences encoding different chimeric adhesins and expressing a mixture of chimeric adhesins. For example, a nucleic acid encoding ErbB2-IgG and a nucleic acid encoding ErbB3-IgG are introduced into a host cell, expressed, and a mixture of the homodimers and heterodimer is isolated from the cell or from the culture medium.
An embodiment of the invention further includes a host cell comprising the nucleic acid of the invention. Preferably the host cell is capable of expressing the nucleic acid, which expression includes the translation and production of the chimeric heteroadhesin of the invention. The embodiment of the invention encompasses a host cell comprising and expressing a chimeric monomer of the heteroadhesin, while in another host cell of the invention an additional chimeric monomer of the heteroadhesin is expressed. Alternatively, the embodiment encompasses the expression of more than one chimeric monomer in a single host cell.
In a preferred embodiment of the invention, the host cell comprises a first isolated nucleic acid sequence encoding the first amino acid sequence of the soluble chimeric heteromultimer of the invention, wherein the extracellular domain is from the ErbB2 receptor and wherein the multimerization domain comprises an immunoglobulin constant region; and a second isolated nucleic acid sequence encoding an additional amino acid sequence of the soluble chimeric heteromultimer of the invention, wherein the extracellular domain is from the ErbB3 receptor and wherein the multimerization domain comprises an immunoglobulin constant region.
In another preferred embodiment of the invention, the host cell comprises a first isolated nucleic acid sequence encoding the first amino acid sequence of the soluble chimeric heteromultimer of the invention, wherein the extracellular domain is from the ErbB2 receptor and wherein the multimerization domain comprises an immunoglobulin constant region; and a second isolated nucleic acid sequence encoding an additional amino acid sequence of the soluble chimeric heteromultimer of the invention, wherein the extracellular domain is from the ErbB4 receptor and wherein the multimerization domain comprises an immunoglobulin constant region.
Another aspect of the invention includes an antagonist antibody to the chimeric heteromultimer adhesin of the invention, wherein the antibody binds to the natural heteromultimeric receptor and inhibits its activation.
Another aspect of the invention includes an agonist antibody to the chimeric heteromultimer adhesin of the invention, wherein the antibody binds to the natural heteromultimeric receptor and activates it. In preferred embodiments of the invention, the agonist antibody is capable of activating the natural heteromultimeric receptor at 10xe2x88x921 to 106 fold the activity of the natural ligand.
The chimeric heteromultimer-specific antibodies may be used, among other things, in a method for detecting heteromultimeric receptors which comprises the step of contacting a sample suspected of containing the heteromultimeric receptor with the antibody (which is optionally labeled) and detecting if binding has occurred. The antibody may also be used in a method for purifying the heteromultimeric receptor which comprises the step of passing a mixture containing the heteromultimeric receptor over a solid phase to which is bound the antibody and recovering the fraction containing the heteromultimeric receptor. Preferably, in one embodiment of the invention the heteromultimeric receptor is ErbB2/ErbB4 and the chimeric heteromultimer adhesin is ErbB2-IgG/ErbB4-IgG. In another preferred embodiment, the heteromultimeric receptor is ErbB2/ErbB3 and the chimeric heteromultimer adhesin is ErbB2-IgG/ErbB3-IgG.
In yet another aspect, the invention includes a method of forming a chimeric heteromultimer adhesin-ligand complex in a sample comprising the ligand. The method of the invention includes contacting the chimeric heteromultimer adhesin of the invention with the sample under conditions such that the ligand binds to the heteromultimer to form a chimeric heterodimer adhesin-ligand complex.
In an embodiment of the invention, the chimeric heteromultimer adhesin-ligand complex inhibits binding of the ligand to the natural heteromultimer receptor. Preferably the sample is a mammalian tissue or a mammalian fluid, such as a body fluid including, but not limited to blood, serum, plasma, lymph, and urine. Preferably, the mammal is a human.
In another aspect, the invention involves a method of inhibiting natural heteromultimer receptor activation. The method includes the steps of 1) contacting the chimeric heteromultimer adhesin of the invention with a sample containing a ligand for the natural heteromultimeric receptor and the receptor; and 2) incubating the chimeric heteromultimer adhesin with the ligand to form a complex such that activation of the natural heteromultimeric receptor by the ligand is inhibited.
In an embodiment of the method of inhibiting ligand binding to a natural heteromultimer receptor, the natural heteromultimeric receptor is ErbB and the soluble chimeric heteromultimer comprises the extracellular domains of ErbB2 and ErbB3.
In another embodiment of the method of inhibiting ligand binding to a natural heteromultimer receptor, the natural heteromultimeric receptor is ErbB and the soluble chimeric heteromultimer comprises the extracellular domains of ErbB2 and ErbB4.
Another embodiment of the invention is a method of inhibiting ligand binding to a natural heteromultimer receptor, wherein receptor activation is inhibited. The method comprises contacting the antagonist antibody of the invention with the natural heteromultimeric receptor to form an antagonist antibody-heteromultimer receptor complex, wherein activation of the receptor is inhibited.
In another aspect, the invention involves a method of activating a natural heteromultimeric receptor comprising contacting the agonist antibody of the invention with the natural heteromultimeric receptor to form agonist antibody-heteromultimeric receptor complex, wherein the receptor is activated.
In still another aspect, the invention involves a method for the treatment of a disease state comprising administering to a mammal in need thereof a therapeutically effective dose of the chimeric heteromultimer adhesin of the invention. Embodiments of the invention encompass disease states in which the disease is treatable by inhibiting contact between the ligand and the natural heteromultimeric receptor such as by competitive binding of the heteroadhesin to the ligand.
In an embodiment of the invention, the chimeric heteromultimer adhesin is an ErbB2/ErbB3-Ig heteroadhesin. In another embodiment, the chimeric heteromultimer is an ErbB2/ErbB4-Ig heteroadhesin.
The invention encompasses a composition comprising the chimeric heteromultimer adhesin. The composition comprising the adhesin is preferably sterile. Where the composition is an aqueous solution, preferably the adhesin is soluble. Where the composition is a lyophilized powder, preferably the powder is reconstitutable in an appropriate solvent.
In another embodiment of the invention, the treatment method comprises administering chimeric heteromultimer adhesins which comprise chimeric monomers, each prepared using an extracellular domain of the heteromultimeric receptor monomers of interest. The extracellular domains are preferably from receptors selected from the following heteromultimeric receptors: Axl, Rse, epidermal growth factor (EGF) receptor, hepatocyte growth factor (HGF) receptor, IL-2, c-mer, Al-1, EPH, TrkA, TrkB, TrkC, TNF, IL-10, CRF2-4, RXR, RON, AChRxcex1/xcex4, TRxcex1/RXRxcex1, Trxcex1/DR4, Trxcex1/MHC-TRE, Trxcex1/ME, Trxcex1/F2, KDR/FLT-1, FLT/VEGF, VEGF121/165, Arnt/Ahr, CGA/CGB, EGFR/p185-neu, prolactin receptor (PRL), T cell receptor (TCR), fibroblast growth factor (FGF), and Cak receptor (Kishimoto, T. et al. (1994) Cell 76:253-262; Kendall, R. L., et al. (1996) Biochem Biophys. Res. Comm. 226:324-328; Chang, W.-P. And Clevenger, C. V. (1996) PNAS USA 93:5947-5952; Lala, D. S. et al. (1996) Nature 383:450-453; Collesi, C. et al. (1996) Mol. Cell. Biol. 16:5518-5526; Tzahar, E. et al. (1996) Mol. Cell. Biol. 16:5276-5287; Shtrom, S. S. and Hall, Z. W. (1996) J. Biol. Chem. 271:25506-25514; Nagaya, T. et al. (1996) Biochem. Biophys. Res. Comm. 226:426-430; Dendall, R. L. et al. (1996) Biochem. Biophys. Res. Comm. 226:324-328; Kainu, T. et al. (1995) Neuroreport 6:2557-2560; Yoo, S. H. and Lewis, M. S. (1996) J. Biol. Chem. 271:17041-17046; Murali, R. et al. (1996) PNAS USA 93:6252-6257; Dietrich J. et al. (1996) J. Cell Biol. 132:299-310; Tanahashi, T. et al. (1996) J. Biol. Chem 271:8221-8227; and Perez, J. L. et al. (1996) Oncogene 12:1469-1477). The extracellular domains are more preferably from receptors selected from the following: IL-6/gp130, IL-11/gp130 leukemia inhibitory factor (LIF)/gp130, cardiotrophin-1/gp130 (CT-1), IL-11/gp130, ciliary neurotrophic factor CNTF/gp130, oncostatin M (OSM)/gp130, interferon xcex3, and interferon xcex1, xcex2 (Kishimoto, T. et al. (1994), supra; Taga, T. (1996) J. Neurochem. 67:1-10; Pennica, D. et al. (1995) J. Biol. Chem. 270:10915-10922; Marsters, S. A. (1995) PNAS USA 92:5401-5405; and Wollert, K. C. et al. (1996) J. Biol. Chem. 271:9535-9545). Most preferably, the extracellular domains are selected from the ErbB family of receptors.
Embodiments of the method of treatment encompass a disease state or states such as immunological disorders, cancer, and neurological disorder.
In embodiments where the heteroadhesin is an ErbB2/ErbB3-Ig or an ErbB2/ErbB4-Ig heteroadhesin, the method of treatment encompasses a disease state selected from the group consisting of inflammatory disease, cancer, neurological disorders such as neurofibromatosis and peripheral neuropathy, and cardiac disorders such as cardiac hypertrophy.
The invention further provides a method for treating a mammal comprising administering a therapeutically effective amount of a chimeric heteromultimer adhesin, such as ErbB2/3-IgG or ErbB2/4-IgG to the mammal. For example, the mammal may be suffering from a neurological disorder or cell proliferative disease. The mammal is one which could benefit from a reduction in HRG levels/biological activity (e.g. in cancer).
In another aspect, the invention includes pharmaceutical compositions. In an embodiment of the invention the pharmaceutical composition comprises a chimeric heteromultimer adhesin of the invention, which heteroadhesin 1) comprises an ECD or binding fragment thereof of a natural heteromultimeric receptor, and 2) is an antagonist of the ligand which binds the ECD of the natural heteromultimeric receptor.
In another embodiment of the invention the pharmaceutical composition comprises an antibody to a chimeric heteromultimer adhesin of the invention, which anti-heteroadhesin antibody 1) comprises an ECD or binding fragment thereof of a natural heteromultimeric receptor, and 2) binds to the ECD of the natural heteromultimeric receptor and is an antagonist of the ligand which binds the ECD of the natural heteromultimeric receptor.
In still another embodiment of the invention the pharmaceutical composition comprises an antibody to a chimeric heteromultimer adhesin of the invention, which anti-heteroadhesin antibody 1) comprises and ECD or binding fragment thereof of a natural heteromultimeric receptor, and 2) binds to the ECD of the natural heteromultimeric receptor and is an agonist of the ligand which binds the ECD of the natural heteromultimeric receptor.
In yet another aspect, the invention includes articles of manufacture comprising a container, a label on the container, and a composition contained within the container. In one embodiment of the invention, the composition comprises the chimeric heteromultimer adhesin composition of the invention, which heteroadhesin is an antagonist of ligand. The composition is effective for antagonizing binding of the ligand to its natural heteromultimeric receptor, and the label on the container indicates that the composition can be used for antagonizing binding of the ligand to the natural heteromultimeric receptor. In a preferred embodiment the chimeric heteromultimer adhesin is selected from the group consisting of ErbB2/ErbB3-Ig or ErbB2/ErbB4-Ig.
In another embodiment of the article of manufacture, the composition comprises an anti-chimeric heteromultimer adhesin antibody, which antibody is an antagonist of a ligand. The composition is effective for antagonizing binding of the ligand to its natural heteromultimeric receptor, and the label on the container indicates that the composition can be used for antagonizing binding of the ligand to the natural heteromultimeric receptor. In a preferred embodiment the anti-chimeric heteromultimer adhesin antibody is an antibody raised to a chimeric heteroadhesin selected from the group consisting of ErbB2/ErbB3-Ig or ErbB2/ErbB4-Ig.
In yet another embodiment of the article of manufacture, the composition comprises an anti-chimeric heteromultimer adhesin antibody, which antibody is an agonist of a ligand. The composition is effective for activating the natural heteromultimeric receptor of the ligand, and the label on the container indicates that the composition can be used for activating the natural heteromultimeric receptor. In a preferred embodiment the anti-chimeric heteromultimer adhesin antibody is an antibody raised to a chimeric heteroadhesin selected from the group consisting of ErbB2/ErbB3-Ig or ErbB2/ErbB4-Ig.
These and other aspects of the invention will be apparent to those skilled in the art upon consideration of the following detailed description.